Direct sandwich ELISA to detect the adulteration of human breast milk by cow milk

The demand for commercially available human breast milk has significantly increased in recent years. For various reasons, a significant amount of commercially available human breast milk is being adulterated with other types of milk. This fraudulent practice poses a threat to consumers' health due to potential adulterants such as cow milk, which may put the infant at risk due to intolerance or allergy. A direct sandwich anti-bovine IgG ELISA has been developed for the sensitive and specific detection of cow milk in adulterated human breast milk. This assay uses polyclonal anti-bovine IgG antibody as a capture antibody and monoclonal anti-bovine IgG-alkaline phosphatase antibody as a detection antibody. Once optimized, the assay was found to be highly sensitive, and specific to bovine IgG. The assay had no significant cross-reaction with human breast milk, indicating that it was highly specific. The anti-bovine IgG ELISA was able to detect the presence of cow milk in adulterated human breast milk with a detection limit of 0.001% cow milk. The developed assay was highly reproducible (coefficient of variation <10%). The developed direct sandwich anti-bovine IgG ELISA is simple, reliable, and reproducible, making it an ideal test for this purpose.     

Indirect ELISA for detection of goats' milk in ewes' milk and cheese

An indirect ELISA (enzyme-linked immunosorbent assay) has been developed successfully for the detection of defined amounts of goats' milk (1–25%) in ewes' milk and cheese. The assay uses polyclonal antibodies raised in rabbits against goats' caseins (GC). The anti-GC antibodies were recovered from the crude antiserum by immunoadsorption and elution from a column containing immobilized goats' caseins. The anti-GC antibodies were biotinylated and rendered goats' milk specific by mixing them with lyophilized bovine and ovine caseins. The assay was developed in a non-competitive ELISA format and it comprised coating plates with extracts from samples. ExtrAvidin-peroxidase was used to detect the biotinylated anti-GC antibodies bound to goats' caseins immobilized on 96-well plates. The colour developed by the subsequent enzymic conversion of the substrate resulted in discernible differences in optical densities when assaying mixtures of ewes' milk and cheese containing variable amounts of goats' milk.     

Rapid detection and quantification of milk adulteration using infrared microspectroscopy and chemometrics analysis

The application of attenuated total reflectance mid-infrared microspectroscopy (MIR-microspectroscopy) was evaluated as a rapid method for detection and quantification of milk adulteration. Milk samples were purchased from local grocery stores (Columbus, OH, USA) and spiked at different concentrations of whey, hydrogen peroxide, synthetic urine, urea and synthetic milk. Samples were place on a 192-well microarray slide, air-dried and spectra were collected by using MIR-microspectroscopy. Pattern recognition analysis by Soft Independent Modeling of Class Analogy (SIMCA) showed tight and well-separated clusters allowing discrimination of control samples from adulterated milk. Partial Least Squares Regression (PLSR) showed standard error of prediction (SEP) ∼2.33, 0.06, 0.41, 0.30 and 0.014 g/L for estimation of levels of adulteration with whey, synthetic milk, synthetic urine, urea and hydrogen peroxide, respectively. Results showed that MIR-microspectroscopy can provide an alternative methodology to the dairy industry for screening potential fraudulent practice for economic adulteration of cow’s milk.     

Measurement of bovine IgG by indirect competitive ELISA as a means of detecting milk adulteration

The aim of this work was to develop an assay capable of detecting adulteration of high premium milk with milk from cheaper sources. An indirect, competitive ELISA was developed for the rapid detection of cows' milk in the milk of goat, sheep, and buffalo. The assay uses a monoclonal antibody produced against bovine IgG. This antibody recognizes a species-specific epitope on the heavy chain of both bovine IgG1 and IgG2. A peroxidase-conjugated anti-mouse IgG antibody was used to detect bound monoclonal antibody and subsequent enzymatic conversion of substrate resulted in clear differences in absorbance when assaying different mixtures of milks adulterated with cows' milk. Once optimized, the ELISA was found to be highly specific. Detection limits of the assay are 1.0 microg/mL of bovine IgG, or 0.1% (vol/vol) adulteration with cows' milk. The assay was highly reproducible (CV < 10%) and performed equally well when used to detect bovine IgG in mixtures with the 3 types of milk tested. The ELISA performance makes it suitable for development as a kit, for use in the field as a high throughput screening ELISA.     

Detection of cows' milk in ewes' milk and cheese by a sandwich enzyme-linked immunosorbent assay (ELISA)

A sandwich enzyme-linked immunosorbent assay (ELISA) has been successfully developed for the detection of defined amounts of cows' milk in ewes' milk and cheese. Polyclonal antibodies were raised in goats against bovine caseins (BC). The resultant antibodies were recovered from the crude antiserum by ammonium sulphate precipitation and further purified by immunoatisorption of the cross-reacting antibodies onto columns containing immobilised ovine, caprine and bovine caseins, followed by elution of the bovine caseins specific antibodies (anti-BC) from the column containing the bovine caseins. The anti-BC bound to the wells of a microtitre plate were used to capture the BC from milk and cheese mixtures. Further immunorecognition of the captured proteins was attained with the same specific antibodies conjugated to biotin. ExtrAvidin-peroxidase was used to detect biotinylated antibodies bound to their specific antigens. Subsequent enzymic conversion of substrate gave clear absorbance differences when assaying mixtures of ewes' milk and cheese containing variable amounts of cows' milk.     

Detection of cow milk adulteration in yak milk by ELISA

In the current study, a simple, sensitive, and specific ELISA assay using a high-affinity anti-bovine β-casein monoclonal antibody was developed for the rapid detection of cow milk in adulterated yak milk. The developed ELISA was highly specific and could be applied to detect bovine β-casein (10–8,000 μg/mL) and cow milk (1:1,300 to 1:2 dilution) in yak milk. Cross-reactivity was <1% when tested against yak milk. The linear range of adulterant concentration was 1 to 80% (vol/vol) and the minimum detection limit was 1% (vol/vol) cow milk in yak milk. Different treatments, including heating, acidification, and rennet addition, did not interfere with the assay. Moreover, the results were highly reproducible (coefficient of variation <10%) and we detected no significant differences between known and estimated values. Therefore, this assay is appropriate for the routine analysis of yak milk adulterated with cow milk.     

双抗体夹心酶联免疫法测定水牛初乳中的IgG

为快速、准确地测定水牛初乳中IgG活性质量浓度,建立了双抗体夹心酶联免疫分析法(ELISA)。结果表明,IgG活性质量浓度的对数值与吸光值存在很好的线性关系,其标准曲线方程式为γ=-0.2907x2+2.0819x+0.8689,相关系数R2=0.9937,线性范围在7.8～1000ng/mL,平均回收率在84.40%～96.71%之间,灵敏度高、重现性好,可用于水牛初乳及其制品中IgG活性质量浓度的检测。     

Evaluation of real-time PCR assays for detection and quantification of fraudulent addition of bovine milk to caprine and ovine milk for cheese manufacture

Many typical Italian cheeses made from ovine milk are certified as Protected Designation of Origin (PDO). Because caprine and ovine milk production is limited, the fraudulent addition of cows' milk is widespread. In addition, some compounds in bovine milk have high allergenic potential; therefore, such fraud also has implications for consumer health. In this study, a real-time polymerase chain reaction (real-time PCR) test was developed to detect and quantify cow's milk in caprine and ovine cheeses, based on two target genes. The mitochondrial Cytochrome b gene (Cytb) of Bos taurus was used to detect and quantify bovine DNA. The nuclear gene myostatin (Myo), nuclear ribosomal gene 18S, or mitochondrial gene 16S were used alternatively as universal reference markers. Caprine (n = 30) and ovine (n = 51) cheese samples were purchased and analyzed and most were shown to be contaminated by bovine milk. Pairwise analysis of quantification data using a Spearmann Rank Correlation test demonstrated a highly significant correlation between data obtained with the different reference assays.     

Effect of proteolysis during Cheddar cheese aging on the detection of milk protein residues by ELISA

Cow milk is a common allergenic food, and cow milk-derived cheese retains an appreciable level of allergenicity. The specific and sensitive detection of milk protein residues in foods is needed to protect milk-allergic consumers from exposure to undeclared milk protein residues contained in foods made with milk or milk-derived ingredients or made on shared equipment or in shared facilities with milk or milk-derived ingredients. However, during cheese ripening, milk proteins are degraded by chymosin and milk-derived and bacterial proteases. Commercial allergen-detection methods are not validated for the detection of residues in fermented or hydrolyzed products. The objective of this research was to evaluate commercially available milk ELISA kits for their capability to detect milk protein residues in aged Cheddar cheese. Cheddar cheese was manufactured at a local dairy plant and was aged at 5°C for 24 mo, with samples removed at various time points throughout aging. Milk protein residues and protein profiles were measured using 4 commercial milk ELISA kits and sodium dodecyl sulfate-PAGE. The ELISA data revealed a 90% loss of milk protein residue signal between the youngest and oldest Cheddar cheese samples (0.5 and 24 mo, respectively). Sodium dodecyl sulfate-PAGE analysis showed protein degradation throughout aging, with the highest level of proteolysis observed at 24 mo. Results suggest that current commercial milk ELISA methods can detect milk protein residues in young Cheddar cheese, but the detection signal dramatically decreases during aging. The 4 evaluated ELISA kits were not capable of detecting trace levels of milk protein residues in aged cheese. Reliable detection of allergen residues in fermented food products is critical for upholding allergen-control programs, maintaining product safety, and protecting allergic consumers. Furthermore, this research suggests a novel use of ELISA kits to monitor protein degradation as an indication of cheese ripening.     

原料乳中铵盐掺假检测方法

通过向原料乳中人为添加定量铵盐测试试剂,研究原料乳中铵盐掺假物质定性检测的方法。实验结果表明,该方法用于检测原料乳中铵盐质量分数在0．025％以上,具有良好的可操作性,同时方法操作简单,结果便于观察,适用于在养殖、收购、乳制品企业等进行原料乳质量验收检验。     

Use of high-pressure-treated milk for the production of reduced-fat cheese

Pasteurized (65°C, 30 min), pressurized (400 MPa, 22°C, 15 min) and pasteurized–pressurized milks were used for reduced-fat (approximately 32% of total solids) cheese production. Pressurization of milk increased the yield of reduced-fat cheese through an enhanced β-lactoglobulin and moisture retention. In addition, pressurisation of pasteurized skim milk improved its coagulation properties. The cheeses made from pasteurized–pressurized and pressurized milks showed a faster rate of protein breakdown than the cheese made from pasteurized milk, that might be mainly attributed to a higher level of residual rennet. Hardness of the experimental cheeses, as determined by both the sensory panel and instrumental analyses, decreased as the moisture content and proteolytic degradation of the cheese increased (pasteurized>pressurized>pasteurized–pressurized). In general terms, pressurization of reduced-fat milk prior to cheese-making improved cheese texture and thus accounted for a higher overall acceptability, except for the cheeses made from pasteurized–pressurized milk at 60 d of ripening, whose acceptability score was adversely affected by bitterness.     

Detection and quantification of goat's cheese in ewe's cheese using a monoclonal antibody and two ELISA formats

A stable hybridoma cell line (B2B) has been produced secreting a monoclonal antibody (MAb) specific for the s α_s2‐casein of goats. The MAb B2B was used in two enzyme‐linked immunosorbent assays (ELISA) formats for the detection and quantification of the presence of goat's cheese in ewe's cheese samples. In the indirect ELISA format the limit of detection was 1–25% (w/w) substitution of ewe's cheese samples by goat's cheese. Afterwards, a competitive indirect ELISA was successfully developed for the detection of 0.5 to 25% (w/w) of goat's cheese in ewe's cheese samples. This competitive indirect ELISA is a very sensitive assay, can be performed in less than 5 h and is not influenced by the ripening process in cheese. © 1999 Society of Chemical Industry     

The use of soybean milk in soft cheese making

A method for making Domiati cheese (soft cheese) from a mixture of soybean milk and whole milk has been reconstructed. The results obtained showed some important differences in cheese characteristics as the result of using soybean milk, particularly in the higher moisture, soluble nitrogen and acidity contents. The main organoleptic property affected by the soymilk was the flavour. This improved on ripening.
A number of changes were observed during ripening. The presence of soymilk resulted in (1) less loss of cheese moisture and consequently cheese weight, (2) increase in the development of titratable acidity and (3) increase in protein breakdown.
It is suggested that the soybean milk activates the lactic acid, producing bacteria and the proteolytic enzymes present in cheese.     

指纹图谱技术在生鲜乳掺假检测中的研究进展

生鲜乳是乳制品行业发展的主要原料,是决定乳制品质量的关键因素。然而近年来国内外在乳制品方面的食品安全事件频发,不法分子通过在生鲜乳中掺入虚假物质以获取经济利益的行为已经成为严重的安全问题,对人们健康以及整个乳制品行业造成不良影响。指纹图谱技术是对通过一定的分析工具产生的图像进行判别的一种检测技术,可以对生鲜乳的掺假进行更灵敏、准确和快速的检测。本文通过对生鲜乳的安全现状进行剖析,总结了电泳法、光谱法、色谱法和电子感官技术法4种指纹图谱技术在牛乳掺假检测中的应用,比较了4种技术的优点和局限性,并对未来的研究方向进行了展望,为提高生鲜乳的品质与安全以及保证消费者健康提供理论依据与参考。     

酶联免疫吸附法测定乳粉中IgG含量

母乳化乳粉中添加保健功能因子人Ig G.用饱和硫酸铵法从健康人血清中制备粗Ig G,经Sephadex G-100柱制备纯Ig G;用纯人Ig G作为抗原,免疫兔子制备兔抗人Ig G抗体.建立乳制品中人Ig G含量酶联免疫吸附测定,最佳包板物浓度为5.25μg/ml,最佳血清效价为11 600,抗血清亲和力为2.40×107,回收率为88%～94%,测定Ig G浓度范围为5.0～160 ng/ml,最小检测限为3.25 ng/ml.     

掺假羊乳及其制品中牛乳的检测技术研究进展

羊乳具有营养价值高、蛋白质组成更接近人乳、脂肪球直径小及致敏性低等优点,更利于人体消化吸收,受到消费者和乳品企业的青睐.近年来我国羊乳产业发展迅速且潜力巨大,但由于受羊乳产量和养殖规模的限制,羊乳价格昂贵,市场中存在羊乳及其制品掺假牛乳的现象,且掺假手段多样,难以辨别.为了保证消费者的健康和权益,保障羊乳市场良性发展,...     

An indirect competitive ELISA for the detection of cows’ milk and caseinate in goats’ and ewes’ milk and cheese using polyclonal antibodies against bovine γ-caseins

 An indirect competitive enzyme-linked immunosorbent assay (ELISA) method was developed for the detection of bovine milk and caseinate in goats’ and ewes’ milk and cheese. Polyclonal antibodies were raised in rabbits and chickens against bovine γ₃-casein. In a first affinity chromatography step, antibodies recognizing caseins were absorbed on bovine casein-Sepharose. From the dialysed eluate, antibodies crossreacting with ewes’ and goats’ milk protein were completely removed by immunoadsorption onto stationary phases containing ovine casein and protein extracted from genuine ewes’ and goats’ milk cheese. The detection limit of the ELISA test was 0.1% and the method was applied successfully during an EU collaborative study of the evaluation of methods for the detection of cows’ milk. Received: 15 February 1996     

乳品中热带假丝酵母菌的双抗夹心ELISA快速检测方法

本研究用于乳品中热带假丝酵母菌的快速检测。采用热带假丝酵母菌超声破碎后的上清蛋白作为免疫原分别免疫新西兰大耳白兔和Hartley豚鼠,获得热带假丝酵母菌的多克隆抗体。以兔抗体作为捕获抗体,豚鼠抗体作为检测抗体,通过矩阵法及正交分析建立乳品中热带假丝酵母菌快速、特异的双抗夹心ELISA检测方法。该方法检出限为98 ng/m L,板内变异系数小于2%,板间变异系数小于6%,特异性及重复性良好。实际样品检测中,通过对乳制品进行滤膜集菌并选择性增菌培养,超声后提取的蛋白上清应用本研究建立的双抗夹心ELISA检测方法,100 CFU/m L热带假丝酵母菌可在22 h内准确检测出阳性反应。